The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference.
Electrophoresis gels have been widely used for the separations of biological macromolecules such as proteins, nucleic acids, and the like. There are essentially two types of gels in use: agarose gels and polyacrylamide gels. Polyacrylamide gels, in general, have higher resolving power than agarose gels. Since gel casting is rather tedious and the quality of handcast gels is inconsistent, there is a need for precast, "ready to use" gels. Generally, precast gels are manufactured and supplied in buffers of pH between 8 and 9. Under these conditions, precast agarose gels are stable, and have a shelf life of one year at 4.degree. C. However, precast polyacrylamide gels are unstable, and depending on use, have a shelf life of only three months at 4.degree. C. As precast polyacrylamide gels age in alkaline conditions (pH above 7), the electrophoretic mobility of biological macromolecules through these gels decreases and the separation resolution deteriorates. The short shelf life of precast polyacrylamide gels is primarily attributed to the hydrolytic degradation of acrylamide moieties in the gel, while the crosslinking units, usually N,N'-methylene bisacrylamide, are relatively stable. Due to the short shelf life of precast polyacrylamide gels, it is difficult for a manufacturer to mass produce and to store large quantities of gels, and it is inevitable that some customers have to throw away some unused but "expired" gels. Therefore, it is highly desirable to have a gel that has a similar resolution to polyacrylamide gel, but a longer shelf life. Since the manufacturing and application of precast polyacrylamide gels are well established, it is even more desirable to have a stable, high resolution gel system that can be manufactured and used in the same manner as polyacrylamide gels.
Recognizing the fact that the short shelf life of precast polyacrylamide gels is due to the hydrolytic degradation of acrylamide moieties in alkaline condition, Takeda et al. (U.S. Pat. No. 5,464,516), Engelhorn et al. (U.S. Pat. No. 5,578,180) and Bjellqvist et al. (WO 96/16724) developed neutral buffer systems to replace the Tris.HCl buffer (pH=8.8) in sodium dodecyl sulfate (SDS) polyacrylamide gels, and indeed improved the shelf life of precast polyacrylamide gels. However, the gel running buffer has to be changed accordingly, and the protein separation patterns that are obtained from these systems are different from traditional SDS polyacrylamide electrophoresis based on the Laemmli system (Laemmli, Nature 277:680-685 (1970)).
Several vinyl-based monomers were proposed to replace acrylamide in the standard polyacrylamide gel system in order to improve gel stability. Shorr and Jain (U.S. Pat. No. 5,055,517) disclosed the use of N-mono- or di-substituted acrylamide monomers, such as N,N'-dimethylacrylamide (DMA), in electrophoresis gels. Although DMA is more stable than acrylamide, DMA is very hydrophobic and is useful in only a limited number of electrophoretic applications, such as for certain types of nucleic acid analyses.
Kozulic and Mosbach (U.S. Pat. No. 5,319,046) disclosed the use of N-acryloyl-tris-(hydroxymethyl)aminomethane (NAT), and Kozulic (U.S. Pat. No. 5,202,007) disclosed the use of sugar-based acrylamide derivatives in electrophoresis gels. Because of the presence of several hydroxyl groups in the monomers, these monomers are extremely hydrophilic. However, Chiari et al (Electrophoresis 15:177-186 (1994)) reported that NAT is less stable than acrylamide. On the basis of molecular modeling, Miertus et al (Electrophoresis 15:1104-1111 (1994)) concluded that, when there are two atoms between the amide linkage and the hydroxyl group (as is the case for NAT, sugar-based acrylamide derivatives, and N-(2-hydroxyethyl)acrylamide), the hydroxyl group facilitates the hydrolysis of amide linkages.
In a series of articles and patent application, Righetti et al. (WO 93/11174; Electrophoresis 15:177-186 (1994); Electrophoresis 16:1815-1829 (1995)) disclosed the use of N-mono- and di-substituted hydroxyethoxyethyl-(meth)acrylamides and their analogs in electrophoresis gels. The formula of the monomers disclosed by Righetti et al. in these references is: ##STR1## N-(Hydroxyethoxyethyl)acrylamide (HEEAA) was identified as the preferred monomer, because of its extreme hydrophilicity and resistance to alkaline hydrolysis.
However, Righetti et al. (WO 97/16462; Electrophoresis 17:723-731 (1996); Electrophoresis 17:732-737 (1996); Electrophoresis 17:738-743 (1996)) subsequently reported that the HEEAA monomer had a peculiar tendency to auto-polymerize during storage as a 50% aqueous solution at 4.degree. C., even in the presence of free radical inhibitor. In view of this auto-polymerization tendency of HEEAA, Righetti et al. disclosed in these references the use of N- mono- and di-substituted hydroxyalkyl-(meth)acrylamides as an alternative in electrophoresis gels. The formula of the monomers disclosed by Righetti et al. in these references is: ##STR2## N-(Hydroxypropyl)acrylamide (HPAA) was claimed by Righetti et al. to be extremely hydrophilic and resistant to alkaline hydrolysis. However, there have been no further reports on HPAA-based gels by Righetti's group or other groups, and there have been no HPAA-based commercial products.
Although N-(2-hydroxyethyl)acrylamide (HEAA) is an analog of the N-(hydroxyalkyl) acrylamides disclosed by Righetti (WO 97/16462), it has never been reported or even mentioned as a monomer for electrophoresis gels. For example, Righetti specifically excludes HEAA in his patent applications and references. This is partially because HEAA was not commercially available, but more importantly, HEAA was believed to be unstable to hydrolysis, like N-acryloyl-tris-(hydroxymethyl)aminomethane (NAT) (Electrophoresis 15:1104-1111 (1994)).
Although several preparation methods for HEAA have been reported in the literature, none of them is satisfactory to provide high-purity HEAA with high yield and easy scale-up ability. Saito et al (Macromolecules 29:313-319 (1996)) described a two-phase method for the preparation of HEAA. The organic phase contains acryloyl chloride and ethyl acetate solvent, and the aqueous phase contains sodium hydroxide and ethanolamine. The product is recovered from the organic phase, and further purified by silica gel chromatography. There are two inherent disadvantages with this method, however. First, HEAA is readily soluble in water, and ethyl acetate extraction is not efficient. Second, it is impractical to produce large quantities of HEAA by silica gel chromatography.
Chen (ACS Symposium Series 322:283-290 (1986)) disclosed a one-phase method in which acryloyl chloride was reacted with two equivalents of ethanolamine in acetonitrile. Although high-yield HEAA can be obtained in acetonitrile solution, no purification method was provided, other than removing acetonitrile by distillation. Removal of acetonitrile in this manner results in some polymerization of the HEAA monomer during purification.
Righetti et al (WO 97/16462; Electrophoresis 17:723-731 (1996)) disclosed another onephase method for the preparation of N-(hydroxyalkyl)acrylamides. They reported that ethanol is the best solvent for this reaction. Since ethanol is reactive towards acryloyl chloride, the reaction has to be conducted between -30.degree. C. and -70.degree. C. Silica gel was also used for further purification.
Murashige and Fujimoto (JP 61-068454 and JP 61-000053) disclosed a method in which N-(hydroxyethyl)acrylamide was prepared by treating ethanolamine with C.sub.1-22 alkyl acrylate or acrylic acid. The monomer was directly converted to its polymer, and no monomer purification method was disclosed.
Thus, there is a need to develop additional hydrophilic monomers for preparing electrophoresis compositions, and particularly electrophoresis gels having the combined properties of hydrolytic stability and high resolution. This need in the art is satisfied by the present invention, as described in further detail below.
There further is a need to develop a method for producing high purity N-(hydroxyethyl)acrylamide (HEAA) and similar hydrophilic monomers simply and on a large scale. This need is satisfied by the present invention, as described in further detail below.